Spliced gene of KSHV/HHV8, its promoter and monoclonal antibodies specific for LANA2

ABSTRACT

This invention provides an isolated nucleic acid which encodes a Kaposi&#39;s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 polypeptide (LANA2) or a fragment thereof and also provides the LANA2 polypeptide. This invention provides an isolated nucleic acid comprising consecutive nucleotides having the sequence of a promoter of Kaposi&#39;s sarcoma-associated herpesvirus latency-associated nuclear antigen 2 transcription. This invention also provides a method of inhibiting p53 mediated apoptosis of a cell and a method of producing an antibody which comprises introducing into a cell a replicable vector of the subject invention.

This application is a divisional of U.S. Ser. No. 09/733,728, filed Dec.8, 2000, now U.S. Pat. No. 6,653,465 B2, issued Nov. 25, 2003, thecontents of which are incorporated herein by reference.

The invention described herein was made with Government support undergrant number R01 CA67391 from the National Cancer Institute.Accordingly, the United States Government has certain rights in thisinvention.

Throughout this application, various publications are referenced withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citations for these references may be found immediatelypreceding the claims.

BACKGROUND OF THE INVENTION

Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8(HHV8) is the most recently described DNA tumor virus. It is theinfectious trigger for Kaposi's sarcoma, body cavity-based primaryeffusion lymphomas (PEL), and some subtypes of multicentric Castleman'sdisease (CD) for review see (37), KSHV-related CD is a polyclonal B cellhyperplasia that is presumably driven by KSHV vIL-6 secretion as well asother viral proteins. In contrast, PEL are B cell lymphomas thatgenerally have a monoclonal origin as determined by immunoglobulin generearrangement and viral terminal repeat analyses (7, 20, 36). Terminalrepeat analyses by Judde and colleagues (20) have also demonstrated thatKS tumors can have an oligo- or monoclonal pattern, and may evolve froma polyclonal hyperplasia into a monoclonal tumor. Thus, KSHV maycontribute to cell proliferation through secretion of viral cytokinesand induction of cellular cytokines as in the case of CD, as well asthrough expression of transforming viral oncogenes, particularly in thecase of PEL.

The KSHV genome has significant sequence homology to all classes ofherpesviruses, but is unique among the human herpesviruses in encodingan extensive number of regulatory genes which have been pirated from thehost genome during its evolution (30, 36). While a number of these geneshave homology to known cellular oncogenes or transform rodent cell linesin vitro (2, 14, 26), only a small number of KSHV genes are routinelyfound to be expressed in tumor tissues. vBCL-2, vIRF1, vGPCR, and K01are examples of KSHV proteins which might contribute to celltransformation in vitro but are not appreciably expressed in mostKSHV-infected KS or PEL tumors (21, 24, 32, 38).

KSHV infected PEL cell lines constitutively express three viral genes,vFLIP (ORFK13), vCYC (ORF72), and LANA1 (ORF73), which are not inducibleby tetradecanoyl phorbol acetate (TPA) or inhibited by phosphonoformicacid (PFA) and thus are unambiguously designated as latent or class Igenes. These three proteins are transcribed on the major polycistroniclatent transcripts, LT1 and LT2 (10, 39, 42). In vitro studiesdemonstrate that the viral cyclin associates with cyclin dependentkinase (CDK) 4 and 6, and phosphorylates pRB (8, 16, 28). LANA1 isbelieved to bind to the origin of replication to tether the viral genometo host chromatin during mitosis, effecting equal segregation of viralgenome during division (3). LANA1 also binds to p53 and inhibitsp53-mediated transcriptional activity and apoptosis (13). vCYCover-expression induces apoptosis (31) and it is at least theoreticallypossible that this may be inhibited in situ by the anti-apoptoticactivities of other latency expressed proteins, such as vFLIP and LANA1.

Viral protein expression is highly restricted in KS and PEL tumors.Presently, only LANA1 protein has been shown by immunohistochemistry tobe expressed in situ in all cells infected by KSHV (11, 22, 32). Viralcyclin and ORFK12 transcripts have been identified by in situhybridization in all KSHV infected cells (9, 34), however, proteinlocalization has yet to be performed. No other viral proteins examinedthus far, including vIL-6 (K2), minor capsid protein (ORF26), K8, K8.1,vIRF1 (K9), K10, K11, PF-8 (ORF59), and ORF65 have a similar in situconstitutive pattern of expression (21, 32).

KSHV gene expression studies remain controversial. Since PEL cell linescan be manipulated into lytic replication by TPA and butyrate, studieson cultured cell lines have been used to classify KSHV genes intomutually-exclusive latent and lytic classes based on transcriptionkinetics (40). Frequently, KSHV expression patterns from cultured cellstudies are assumed to be similar in tumor tissues in situ withoutdirect evidence. However, a number of KSHV genes are expressed at lowlevels in resting PEL cell lines but are induced to high expressionlevels during TPA treatment and thus have properties of both latent andlytic genes (analogous to the EBV LMP1 expression pattern). This patternof gene expression has been referred to as class II expression (37).Recent studies demonstrate that extension of results from expressionstudies in tissue culture cannot be uniformly applied to human tumortissues in part because KSHV may have tissue-specific gene expressionpatterns. vIL-6, for example, behaves as a class II protein in tissueculture cell lines and is expressed in hematopoietic-derived cells butgenerally not in KS lesions (29). Thus, determining precisely whichviral genes are likely to play a role in KSHV-related pathogenesisrequires direct tissue examination of each tumor type. <Discovery ofadditional genes that are constitutively expressed in KSHV-induceddisorders is particularly important since these genes are likely to playa role in cell growth dysregulation.

For these reasons, discovery of a KSHV gene having a tissue-specificexpression profile is important, particularly if the encoded protein isfunctionally capable of contributing to cell proliferation. In thispaper we describe a new KSHV gene (K10.5) expressed in KSHV-infectedhematopoietic tissues. This gene is located in a region containing acluster of viral sequences with limited homology to the interferonregulatory factor (IRF) family of proteins (36). vIRF1 is encoded by ORFK9 and inhibits interferon-induced transcription and fully transformsNIH3T3 cells (12, 14, 27, 44). vIRF1 binds to histone acetyltransferasetranscriptional coadaptors (5, 19) and induces cell transformation byactivating the cMYC oncogene through an interferon-stimulated responseelement (ISRE) called the PRF element (19). Based on these findings andthe fact that other tumor viruses target the same tumor suppressorpathways as KSHV, Jayachandra et al. found that both Epstein-Barr virus(EBV or HHV4) EBNA2 and adenovirus E1A proteins also activate cMYC butuse differing sets of coadaptors from those used by vIRF1 (19). vIRF1additionally inhibits p53- and Fas-induced apoptosis ((5) andunpublished obs, S. Jayachandra, P. S. Moore, Y. Chang). vIRF1; however,is not generally expressed in PEL or KS and is therefore unlikely tocontribute to these diseases although it may be important in thepathogenesis of CD (21, 32). Another IRF-like KSHV open reading frameencoding vIRF2 and having NF-kB-inhibitory activity has been described(6). We show here that LANA2 is a B-cell specific factor thatantagonizes p53 tumor suppressor functions and is expressed duringlatency.

KSHV/HHV8 is associated with three proliferative diseases ranging fromviral cytokine-induced hyperplasia to monoclonal neoplasia: multicentricCastleman's disease (CD), Kaposi's sarcoma (KS), and primary effusionlymphoma (PEL).

Here we report a new latency-associated 1704 bp KSHV spliced genebelonging to a cluster of KSHV sequences having homology to theinterferon regulatory factor (IRF) family of transcription factors.ORFK10.5 encodes a protein, latency-associated nuclear antigen 2(LANA2), which is expressed in KSHV-infected hematopoietic tissuesincluding PEL and CD, but not KS lesions. LANA2 is abundantly expressedin the nuclei of cultured KSHV infected B-cells. Transcription of K10.5in PEL cell cultures is not inhibited by DNA polymerase inhibitors norsignificantly induced by phorbol ester treatment. Unlike LANA1, LANA2does not elicit a serologic response from patients with KS, PEL or CD asmeasured by Western-blot hybridization. Both KSHV vIRF1 (ORFK9) andLANA2 (ORFK10.5) appear to have arisen through gene duplication of acaptured cellular IRF gene. LANA2 is a potent inhibitor of p53-inducedtranscription in reporter assays. LANA2 antagonizes apoptosis due to p53overexpression in p53-null SAOS-2 cells and apoptosis due to doxorubicintreatment of wild-type p53 U2OS cells. While LANA2 specificallyinteracts with aminoacids 290-393 of p53 in glutathione-S-transferasepull-down assays, we were unable to demonstrate LANA2-p53 interaction invivo by immunoprecipitation. These findings show that KSHV hastissue-specific latent gene expression programs and identify a newlatent protein which may contribute to KSHV tumorigenesis inhematopoietic tissues via p53 inhibition.

SUMMARY OF THE INVENTION

This invention provides an isolated nucleic acid which encodes aKaposi's sarcoma-associated herpesvirus latency-associated nuclearantigen 2 polypeptide (LANA2) or a fragment thereof.

This invention provides a replicable vector which comprises the isolatednucleic acid which encodes a Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 polypeptide.

This invention provides a host vector system which comprises the abovevector and a suitable host cell. In one embodiment of the above hostvector system, the host cell includes but is not limited to a eukaryoticcell, a hematopoietic cell, a B cell, a bacterial cell and E. Coli.

This invention provides a method of producing a polypeptide whichcomprises growing the above host vector system under suitable conditionspermitting production of the polypeptide and recovering the polypeptideso produced.

This invention further provides an isolated nucleic acid comprisingconsecutive nucleotides having the sequence of a promoter of Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2transcription.

This invention provides a replicable vector which comprises the isolatednucleic acid comprising consecutive nucleotides having the sequence of apromoter of latency-associated nuclear antigen 2 transcription operablylinked to a second nucleic acid.

This invention provides a host vector system which comprises areplicable vector which comprises the nucleic acid comprisingconsecutive nucleotides having the sequence of a promoter oflatency-associated nuclear antigen 2 transcription operably linked to asecond nucleic acid and a suitable host cell.

This invention provides a method of producing a polypeptide whichcomprises growing the above host vector system under suitable conditionspermitting production of the polypeptide and recovering the polypeptideso produced.

This invention provides an isolated nucleic acid capable of specificallyhybridizing to the isolated nucleic acid which encodes a Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide or a fragment thereof.

This invention provides a Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 polypeptide or a fragment thereof.

This invention also provides an antibody capable of specifically bindingto the above polypeptide.

This invention provides a composition comprising the above antibody andan agent conjugated to the antibody.

This invention provides a method of determining whether a subject isafflicted with a disease associated with Kaposi's sarcoma-associatedherpesvirus (KSHV) infection of a B cell which comprises: (a) obtaininga suitable sample from the subject; (b) contacting the suitable samplewith a detectable antibody capable of binding to Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide or a fragment thereof so as to form a complex between theantibody and any Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 polypeptide or a fragment thereofpresent in the sample; (c) removing any unbound antibody; and (d)detecting any antibody which is bound to any Kaposi's sarcoma-associatedherpesvirus latency-associated nuclear antigen 2 polypeptide or afragment thereof in the sample, wherein the presence of antibodyindicates that the subject is afflicted with the disease associated withKaposi's sarcoma-associated herpesvirus infection of a B cell.

This invention provides a method of determining whether a subject isafflicted with a disease associated with Kaposi's sarcoma-associatedherpesvirus infection of a B cell which comprises: (a) obtaining asuitable sample from the subject; (b) immobilizing a capturing antibodywherein the capturing antibody is capable of binding to Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2 to asupport; (c) removing any unbound capturing antibody; (d) contacting thecapturing antibody with the suitable sample so as to form a complexbetween the antibody and any Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 present in the sample; (e) removingany unbound sample; (f) contacting the complex obtained in step (d) witha detectable antibody of Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 polypeptide or a fragment thereofso as to form a complex between the detectable antibody and the complex;(g) removing any unbound detectable antibody; and (h) detecting anydetectable antibody which is bound to the complex wherein the presenceof detectable antibody indicates that the subject is afflicted with thedisease associated with Kaposi's sarcoma-associated herpesvirusinfection of a B cell.

This invention provides a method of determining whether a subject isinfected with Kaposi's sarcoma-associated herpesvirus which comprises:(a) obtaining a suitable sample from the subject; (b) contacting thesuitable sample with the detectable antibody of Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide or a fragment thereof so as to form a complex between theantibody and any polypeptide or fragment thereof present in the sample;(c) removing any unbound antibody; and (d) detecting any antibody whichis bound to any Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 polypeptide or fragment thereof inthe sample, wherein the presence of antibody indicates that the subjectis infected with Kaposi's sarcoma-associated herpesvirus.

This invention provides a method of determining whether a subject isinfected with Kaposi's sarcoma-associated herpesvirus which comprises:(a) obtaining a suitable sample from the subject; (b) immobilizing acapturing antibody wherein the capturing antibody is capable of bindingto polypeptide or fragment thereof to a support; (c) removing anyunbound capturing antibody; (d) contacting the capturing antibody withthe suitable sample so as to form a complex between the antibody andKaposi's sarcoma-associated herpesvirus latency-associated nuclearantigen 2 polypeptide or fragment thereof present in the sample; (e)removing any unbound sample; (f) contacting the complex obtained in step(d) with the detectable antibody which is bound to Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide or fragment thereof so as to form a complex between thedetectable antibody and the complex; (g) removing any unbound detectableantibody; and (h) detecting any detectable antibody which is bound tothe complex wherein the presence of detectable antibody indicates thatthe subject is infected with Kaposi's sarcoma-associated herpesvirus.

This invention provides a kit for diagnosing Kaposi's sarcoma-associatedherpesvirus infection comprising the labeled antibody capable ofspecifically binding to the Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 polypeptide or fragment thereof.

This invention provides a method of inhibiting p53 mediated apoptosis ofa cell which comprises introducing into the cell an effective amount ofthe replicable vector which comprises the isolated nucleic acid whichencodes Kaposi's sarcoma-associated herpesvirus latency-associatednuclear antigen 2 polypeptide or fragment thereof, so as to therebyinhibit p53 mediated apostosis of the cell.

This invention provides a method of immortalizing a cell which comprisesintroducing into the cell an amount of the replicable vector whichcomprises the isolated nucleic acid which encodes Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide or fragment thereof effective to inhibit p53 mediatedapoptosis of the cell, so as to thereby immortalize the cell.

This invention provides a method of producing an antibody whichcomprises introducing into a cell an amount of the replicable vectorwhich comprises the isolated nucleic acid which encodes Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide effective to inhibit p53 mediated apoptosis of the cellproducing the antibody and thereby immortalizing the cell, so as tothereby produce the antibody.

This invention provides a method of determining whether a subject isinfected with Kaposi's sarcoma-associated herpesvirus which comprises:(a) obtaining a suitable sample from the subject; (b) contacting thesuitable sample with a detectable nucleic acid capable of hybriding to anucleic acid which encodes Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 polypeptide or fragment thereofunder hybridizing conditions so as to form a complex between thedetectable nucleic acid and any nucleic acid which encodes a Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide or fragment thereof which is present in the sample; (c)removing any unbound detectable nucleic acid; and (d) detecting anydetectable nucleic acid which is bound to the complex, wherein thepresence of detectable nucleic acid indicates that the subject isinfected with Kaposi's sarcoma-associated herpesvirus.

This invention provides a kit for diagnosing Kaposi's sarcoma-associatedherpesvirus infection comprising a labeled nucleic acid which encodes aKaposi's sarcoma-associated herpesvirus latency-associated nuclearantigen 2 polypeptide or fragment thereof.

This invention provides a transgenic non-human animal which has stablyintegrated into the genome of its germ cells or somatic cells anexogenous nucleic acid construct wherein the nucleic acid constructcomprises a B-cell specific promoter of Kaposi's sarcoma-associatedherpesvirus latency-associated nuclear antigen 2 operably linked to asecond nucleic acid which encodes a gene of interest and is introducedinto the transgenic non-human animal, or an ancestor, at an embryonicstage. In one embodiment of the above transgenic animal, the animal is amammal.

This invention provides a method for evaluating in a non-humantransgenic animal the potential therapeutic effect of an agent fortreating Kaposi's sarcoma-associated herpesvirus infection in a human,which comprises: (a) providing an agent to a transgenic non-human animalwhose cells comprise the nucleic acid which encodes a Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide; and (b) determining the therapeutic effect of the agent onthe transgenic non-human animal by monitoring Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2expression, wherein a decrease in Kaposi's sarcoma-associatedherpesvirus latency-associated nuclear antigen 2 indicates that theagent would have a potential therapeutic effect on Kaposi'ssarcoma-associated herpesvirus infection in a human. In one embodimentof the above method, the animal is a mammal.

This invention provides a method of treating Kaposi's sarcoma-associatedherpesvirus infection in a subject, which comprises introducing into thesubject's cells an effective amount of the nucleic acid capable ofspecifically hybridizing to the isolated nucleic acid which encodesKaposi's sarcoma-associated latency-associated nuclear antigen 2polypeptide or fragment thereof to hybridize to any of the above nucleicacid which is present in the subject's cells, so as to thereby treatKaposi's sarcoma-associated herpesvirus infection.

This invention provides a method of treating Kaposi's sarcoma-associatedherpesvirus infection in a subject, which comprises introducing into thesubject's cells an effective amount of a nucleic acid capable ofspecifically hybridizing to an isolated nucleic acid comprisingnucleotides having the sequence of a promoter of latency-associatednuclear antigen 2 transcription to hybridize to any of this nucleic acidwhich is present in the subject's cells, so as to thereby treat thesubject.

This invention provides a composition comprising the antibody capable ofspecifically binding to the polypeptide encoded by the isolated nucleicacid which encodes Kaposi's sarcoma-associated latency-associatednuclear antigen 2 polypeptide and a carrier.

This invention provides a method of treating a subject infected withKaposi's sarcoma-associated herpesvirus, which comprises administeringto the subject an amount of the above composition under conditions suchthat the antibody binds to any LANA2 present in the subject, so as tothereby treat the subject.

This invention provides a composition comprising the polypeptide encodedby the isolated nucleic acid which encodes Kaposi's sarcoma-associatedlatency-associated nuclear antigen 2 polypeptide or fragment thereof anda carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

Northern hybridization of BC-1 mRNA with ORF K10.5 probe. Probehybridization for mRNA from uninduced BC-1 cells (lane 1), BC-1 cellstreated with 20 ng/ml TPA for 48 hr (lane 2), BC-1 cells treated with0.5 mM PFA (lane 3), BC-1 cells treated with both 20 ng/ml TPA and 0.5mM PFA (lane 4), and KSHV-negative, EBV-infected P3HR1 cells treatedwith 20 ng/ml TPA (lane 5) are shown. Probe hybridizes to a 1.8 kb bandwhich is not induced by TPA nor inhibited by PFA treatment, consistentwith a latent pattern of viral gene transcription. Same blot is strippedand re-probed with b-actin as a control for loading.

FIG. 2

Transcript map of LANA2 in KSHV genomic environs showing V1 (vIRF1), V2,V3, and V4 probes used for northern blot hybridization. The V3 probe wasused to screen a TPA-induced BC-1 cDNA library. Six phages f672, f701,f702, f703, f731, 741) were isolated containing inserts of variablesizes. One full-length 1735 bp cDNA starting at nt 91,425 andterminating at nt 89,599 was identified and sequenced from phage f703.This cDNA contained a start ATG at position 91,393 and a splicedonor/acceptor site corresponding to nt 90,938/90,837.

FIG. 3

Panel A. Comparison of motifs domains between IRF4/Pip and LANA2. IRF4encodes a 450 aa protein with an N-terminal DNA-binding domain (DBD)defined by five tryptophan residues. This characteristic is not found inLANA2 (567 aa), however, a 213 bp region of LANA2, between amino acid432 and 503, shows 32% amino acid identity with the C-terminalinteraction domain (IAD) of IRF4.

Panel B. Phylogenetic tree for KSHV and human IRF proteins. LANA2 ismost closely related to vIRF1 and vIRF2 suggesting a common origin froman ancestral IRF-like gene. Amino acid sequences were aligned usingclustalW and the phylogenetic tree was generated using the Bootstrap NJtree 1000 program. Protein peptides sources are as follows: hIRF1[gi:87992], hIRF2 [gi:539621], hIRF3 [gi:4504725], hIRF4 [gi:2497445],hIRF5 [gi:4504727], hIRF6 [gi:3122293], hIRF7 [gi:4809288], ICSBP(hIRF8)[gi6016308], ISGF3g [gi:266392], KSHV vIRF1 [gi:4929348], KSHV vIRF2[3152728] and KSHV LANA2 [A4008303]. A phylogenetic tree comparing theIRF-like proteins from the RRV26-95 isolate and the KSHV IRF-likeproteins has been published by Alexander et al (1).

FIG. 4

Cytospin preparation of TPA stimulated BCBL-1 cells immunostained withCM-10A2 mouse monoclonal antibody against LANA2. LANA2 demonstrates afinely speckled nuclear pattern exclusive of nucleolar zones inessentially all BCBL-1 cells (60× magnification, hematoxylincounterstain).

FIG. 5

Immunofluorescence double co-localization of LANA1 and LANA2 in KSHVinfected BCBL-1 cells. Panel A shows LANA1 protein (red) in a coarselyspeckled nuclear distribution, panel B demonstrates the diffuse, finelyspeckled nuclear pattern of LANA2 protein (green), and panel C showsdouble filter, co-localization of the two. Although some subnuclearregions show the distinct dispersal of the two proteins exclusive ofeach other, yellow nuclear staining is also evident in other areaspossibly representing co-localization of a subtraction of LANA1 andLANA2. Cells undergoing mitosis (arrow) appear to express only LANA1exclusive of LANA2 (panel C) (100×, Texas Red and FITC).

FIG. 6

Immunolocalization of LANA1 compared with LANA2 in KSHV-infecteddisorders. Panels A, B, and C represent LANA1 immunolocalization in apericardial PEL infiltrating cardiac muscle, a germinal center from alymph node with multicentric Castleman's disease and a cutaneous KSlesion biopsy, respectively. Adjacent sections of the same tissues areimmunostained for LANA2 in panels D, E, and F. All tumor cells in PELsexpress both LANA1 (A) and LANA2 (D) and the majority of theKSHV-infected mantle zone lymphocytes in CD express both LANA1 (B) andLANA2 (E). However, while the majority of KS spindle cells express LANA1(C), none express LANA2 (F).

FIG. 7

Immunolocalization of LANA1 (panel A) compared with LANA2 (panel B) in alymph node with CD as well as KS. While the KS spindle cells (areawithin guide lines) and some of the mantle zone lymphocytes show strongnuclear positivity to LANA1, the adjacent section immunostained withLANA2 only shows this protein expressed in the lymphocyte subpopulationof KSHV infected cells in the mantle. The CD serves as an internalpositive control for the negative LANA2 immunostaining of KS spindlecells.

FIG. 8

Inhibition of p53 transcriptional activity by LANA2. Representativeluciferase assay showing inhibition of reporter gene expression bytransient transfection of pcDNA.LANA2: A. SAOS-2 cells were transfectedwith 2 mg of plasmid pG13-Luc reporter plasmid together with 0.0 or 0.5mg of pcDNA.p53 and 0.5 or 1 mg pcDNA.LANA2 as indicated. For control,SAOS-2 cells were transfected with the reporter plasmid pGL3-control and0.0, 0.5 or 1 mg of pcDNA.LANA2. B. U2OS cells were transfected with 2mg of plasmid pG13-Luc reporter plasmid with or without 0.5 or 1 mgpcDNA.LANA2 and treated with 0.4 mM Doxorubicin.

FIG. 9

In vitro GST pull down assays using [³⁵S]methionine labeled LANA2 orp53. LANA2 interacts with full length p53 protein as well as the p53region between 290-393 aa

FIG. 10

LANA2 inhibits p53-induced apoptosis. SAOS-2 cells were transfected withpEGFP—F* and the empty expression vector pCDNA3.1 (A), pCDNA.p53 (B) orpCDNA.p53 and pCDNA.LANA2 (C). Total DNA in all transfections wasnormalized using empty expression vector. After 48 h, cells were fixedand stained with propidium iodide. Cellular DNA content was analyzed byflow cytometry. U2OS cells were transfected with pEGFP—F* and the emptyexpression vector pcDNA (D and E) or pcDNA.LANA2 (F). 18 h aftertransfection cells were treated with doxorubicin (0.4 uM) (E and F) andthe cells were processed for DNA content analysis 30 h post treatment.Numbers indicate the percentage of cells in the sub-G1 phase of the cellcycle.

FIG. 11

Negative sero-reactivity to LANA2 in patients with KS, PEL, and MCD.LANA2 expressed in COS7 cells failed to react on western blotting withserum from patients with various KSHV-related disorders. None of 14 serafrom individuals with AIDS-KS (n=4), classical KS (n=4), KSHVseropositive Castleman's disease (n=4) or PEL (n=2) showed serologicreactivity to LANA2. Negative control sera from four blood donors(seronegative for ORF65 and LANA1 antigens) were also non-reactive,whereas the supernatants from two mouse monoclonal LANA2 hybridomaclones (CM-10A2 and CM-8B6) were positive. “1” denotes clones CM-10A2and CM-8B6.

FIG. 12

Kaposi's sarcoma-associated herpesvirus latency-associated nuclearantigen 2 (LANA2) nucleic acid sequence (SEQ ID NO: 1).

FIG. 13

Kaposi's sarcoma-associated herpesvirus latency-associated nuclearantigen 2 (LANA2) polypeptide sequence (SEQ ID NO: 2).

FIG. 14

Kaposi's sarcoma-associated herpesvirus latency-associated nuclearantigen 2 (LANA2) promoter nucleic acid sequence (SEQ ID NO: 3).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following standard abbreviations are used throughoutthe specification to indicate specific amino acids:

A = ala = alanine R = arg = arginine N = asn = asparagine D = asp =aspartic acid C = cys = cysteine Q = gln = glutamine E = glu = glutamicacid G = gly = glycine H = his = histidine I = ile = isoleucine L = leu= leucine K = lys = lysine M = met = methionine F = phe = phenylalanineP = pro = proline S = ser = serine T = thr = threonine W = trp =tryptophan Y = tyr = tyrosine V = val = valine B = asx = asparagine oraspartic acid Z = glx = glutamine or glutamic acid

As used herein, the following standard abbreviations are used throughoutthe specification to indicate specific nucleotides: C=cytosine;A=adenosine; T=thymidine; G=guanosine; and U=uracil.

This invention provides an isolated nucleic acid which encodes aKaposi's sarcoma-associated herpesvirus latency-associated nuclearantigen 2 polypeptide (LANA2) or a fragment thereof.

In one embodiment of the above nucleic acid, the polypeptide comprisesconsecutive amino acids having the amino acid sequence set forth in SEQID NO: 2. In a further embodiment of the above nucleic acid, theisolated nucleic acid is designated ORFK10.5 and comprises consecutivenucleotides having the sequence set forth in SEQ ID NO: 1.

As used herein, the term “nucleic acid” refers to either DNA or RNA,including complementary DNA (cDNA), genomic DNA and messenger RNA(mRNA). As used herein, “genomic” means both coding and non-codingregions of the isolated nucleic acid molecule. “Nucleic acid sequence”refers to a single- or double-stranded polymer of deoxyribonucleotide orribonucleotide bases read from the 5′ to the 3′ end. It includes bothreplicating vectors, infectious polymers of DNA or RNA and nonfunctionalDNA or RNA.

The nucleic acids of the subject invention also include nucleic acidscoding for polypeptide analogs, fragments or derivatives which differfrom the naturally-occurring forms in terms of the identity of one ormore amino acid residues (deletion analogs containing less than all ofthe specified residues; substitution analogs wherein one or moreresidues are replaced by one or more residues; and addition analogs,wherein one or more resides are added to a terminal or medial portion ofthe polypeptide) which share some or all of the properties of thenaturally-occurring forms.

As used herein, the phrase “nucleic acid encoding” refers to a nucleicacid which directs the expression of a specific polypeptide. The nucleicacid sequences include both the DNA strand sequence that is transcribedinto RNA, the complementary DNA strand, and the RNA sequence that istranslated into protein. The nucleic acid includes both the full lengthnucleic acid sequence as well as non-full length sequences. It beingfurther understood that the sequence includes the degenerate codons ofthe native sequence or sequences which may be introduced to providecodon preference in a specific host cell.

As used herein, “peptide” and “polypeptide” are used to denote two ormore amino acids linked by a peptidic bond between the α-carboxyl groupof one amino acid and the α-amino group of the next amino acid. Peptideincludes not only the full-length protein, but also partial-lengthfragments. Peptides may be produced by solid-phase synthetic methodsthat are well-known to those skilled in the art. In addition to theabove set of twenty-two amino acids that are used for protein synthesisin vivo, peptides may contain additional amino acids, including but notlimited to hydroxyproline, sarcosine, and γ-carboxyglutamate. Thepeptides may contain modifying groups including but not limited tosulfate and phosphate moieties. Peptides can be comprised of L- orD-amino acids, which are mirror-image forms with differing opticalproperties. Peptides containing D-amino acids have the advantage ofbeing less susceptible to proteolysis in vivo.

Peptides may by synthesized in monomeric linear form, cyclized form oras oligomers such as branched multiple antigen peptide (MAP) dendrimers(Tam et al. Biopolymers 51:311, 1999). Nonlinear peptides may haveincreased binding affinity by virtue of their restricted conformationsand/or oligomeric nature. Peptides may also be produced usingrecombinant methods as either isolated peptides or as a portion of alarger fusion protein that contains additional amino acid sequences.

Peptides may be chemically conjugated to proteins by a variety ofwell-known methods. Such peptide-protein conjugates can be formulatedwith a suitable adjuvant and administered parenterally for the purposesof generating polyclonal and monoclonal antibodies to the peptides ofinterest. Alternatively, unconjugated peptides can be formulated withadjuvant and administered to laboratory animals for the purposes ofgenerating antibodies. Methods for generating and isolating suchantibodies are well-known to those skilled in the art.

The nucleic acids of the subject invention include but are not limitedto DNA, RNA, mRNA, synthetic DNA, genomic DNA, and cDNA.

In one embodiment, the above nucleic acid is detectable. In oneembodiment of the above nucleic acid, the nucleic acid is labeled with adetectable marker. As used herein, “detectable marker” includes but isnot limited to a radioactive label, or a calorimetric, a luminescent, ora fluorescent marker. As used herein, “labels” include radioactiveisotopes, fluorescent groups and affinity moieties such as biotin thatfacilitate detection of the labeled peptide. Other labels and methodsfor attaching labels to compounds are well-known to those skilled in theart.

This invention provides a replicable vector which comprises the isolatednucleic acid which encodes a Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 polypeptide. In one embodiment, theabove vector includes but is not limited to plasmid, cosmid, λ phage andYAC. As used herein, the term “vector” refers to viral expressionsystems, autonomous self-replicating circular DNA (plasmids), andincludes both expression and nonexpression plasmids. Where a recombinantmicroorganism or cell culture is described as hosting an “expressionvector,” this includes both extrachromosomal circular DNA and DNA thathas been incorporated into the host chromosome(s). Where a vector isbeing maintained by a host cell, the vector may either be stablyreplicated by the cells during mitosis as an autonomous structure, or isincorporated within the host's genome.

This invention provides a host vector system which comprises the abovevector and a suitable host cell. In one embodiment of the above hostvector system, the host cell includes but is not limited to a eukaryoticcell, a hematopoietic cell, a B cell, a bacterial cell and E. Coli.

This invention provides a method of producing a polypeptide whichcomprises growing the above host vector system under suitable conditionspermitting production of the polypeptide and recovering the polypeptideso produced.

This invention further provides an isolated nucleic acid comprisingconsecutive nucleotides having the sequence of a promoter oflatency-associated nuclear antigen 2 transcription. In one embodiment ofthis nucleic acid, the nucleic acid comprises consecutive nucleotideshaving the sequence set forth in SEQ ID NO: 3. The promoter of thesubject invention is capable of driving the expression of any gene in aB cell. Accordingly, this permits one skilled in the art to study geneexpression in certain cells, such as B cells, since there will beexpression of the protein in the B cell. In a transgenic animal, thegene would be expressed in the B cells of the animal.

This invention provides a replicable vector which comprises the isolatednucleic acid comprising consecutive nucleotides having the sequence of apromoter of latency-associated nuclear antigen 2 transcription operablylinked to a second nucleic acid. This second nucleic acid is one whichencodes a protein or gene of interest. As used herein, the term“operably linked” refers to linkage of a promoter upstream from a DNAsequence such that the promoter mediates transcription of the DNAsequence.

The vector of the subject invention includes but is not limited to aplasmid, cosmid, λ phage and YAC.

This invention provides a host vector system which comprises areplicable vector which comprises the nucleic acid comprisingconsecutive nucleotides having the sequence of a promoter oflatency-associated nuclear antigen 2 transcription operably linked to asecond nucleic acid and a suitable host cell. The host cell includes butis not limited to a eukaryotic cell, a hematopoietic cell, a B cell, abacterial cell and E. Coli.

This invention provides a method of producing a polypeptide whichcomprises growing the above host vector system under suitable conditionspermitting production of the polypeptide and recovering the polypeptideso produced.

This invention provides an isolated nucleic acid capable of specificallyhybridizing to the isolated nucleic acid which encodes a Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide or a fragment thereof. This invention also provides anucleic acid capable of specifically hybridizing to the isolated nucleicacid comprising nucleotides having the sequence of a promoter oflatency-associated nuclear antigen 2 transcription. The above nucleicacids include but are not limited to DNA, RNA, mRNA, synthetic DNA,genomic DNA, and cDNA. The phrase “specifically hybridizing” and thephrase “selectively hybridizing” describe a nucleic acid thathybridizes, duplexes or binds only to a particular target DNA or RNAsequence when the target sequences are present in a preparation of totalcellular DNA or RNA. By selectively hybridizing it is meant that anucleic acid binds to a given target in a manner that is detectable in adifferent manner from non-target sequence under high stringencyconditions of hybridization. “Complementary”, “antisense” or “target”nucleic acid sequences refer to those nucleic acid sequences whichselectively and specifically hybridize to a nucleic acid. Properannealing conditions depend, for example, upon a nucleic acid's length,base composition, and the number of mismatches and their position on thenucleic acid, and must often be determined empirically. For discussionsof nucleic acid design and annealing conditions for hybridization, see,for example, Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2nd ed.), Cold Spring Harbor Laboratory, Vols. 1-3 or Ausubel,F., et al. (1987) Current Protocols in Molecular Biology, New York. Theabove hybridizing nucleic acids may vary in length. The hybridizingnucleic acid length includes but is not limited to a nucleic acid of atleast 15 nucleotides in length, of at least 25 nucleotides in length, orat least 50 nucleotides in length.

This invention provides a Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 polypeptide or a fragment thereof.This invention also provides a purified and/or an isolated polypeptide.In one embodiment of the above polypeptide, the nucleic acid comprisesconsecutive nucleotides having the sequence set forth in SEQ ID NO: 1.This invention provides an isolated polypeptide comprising consecutiveamino acids having the amino acid sequence set forth in SEQ ID NO:2.

The phrase “isolated” or “purified” when referring to a polypeptide,means a composition which is essentially free of other cellularcomponents. It is preferably in a homogeneous state although it can bein either a dry or aqueous solution. Purity and homogeneity aretypically determined using analytical chemistry techniques such aspolyacrylamide gel electrophoresis or high performance liquidchromatography. A protein which is the predominant species present in apreparation is substantially purified. Generally, a substantiallypurified or isolated protein will comprise more than 80% of allmacromolecular species present in the preparation. Preferably, theprotein is purified to represent greater than 90% of all macromolecularspecies present. More preferably the protein is purified to greater than95%, and most preferably the protein is purified to essentialhomogeneity, wherein other macromolecular species are not detected byconventional techniques.

This invention also provides an antibody capable of specifically bindingto the above polypeptide. The antibody includes but is not limited to amonoclonal antibody or a polyclonal antibody. The polyclonal andmonoclonal antibodies of the invention are immunoreactive with thepeptides or immunogenic fragments of the peptides or functionallycapable of binding an epitopic determinant of the peptides.

As used herein, “antibody” means an immunoglobulin molecule comprisingtwo heavy chains and two light chains and which recognizes an antigen.The immunoglobulin molecule may derive from any of the commonly knownclasses, including but not limited to IgA, secretory IgA, IgG and IgM.IgG subclasses are also well known to those in the art and include butare not limited to human IgG1, IgG2, IgG3 and IgG4. It includes, by wayof example, both naturally occurring and non-naturally occurringantibodies. Specifically, “antibody” includes polyclonal and monoclonalantibodies, and monovalent and divalent fragments thereof. Furthermore,“antibody” includes chimeric antibodies, wholly synthetic antibodies,single chain antibodies, and fragments thereof. The antibody may be ahuman or nonhuman antibody. A nonhuman antibody may be humanized byrecombinant methods to reduce its immunogenicity in man.

The phrase “specifically binding” refers to a binding reaction which isdeterminative of the presence of the LANA2 polypeptide of the inventionin the presence of a heterogeneous population of polypeptides and otherbiologics including viruses other than KSHV. Thus, under designatedimmunoassay conditions, the specified antibodies bind to the LANA2antigen and do not bind in a significant amount to other antigenspresent in the sample.

In one embodiment of the above antibody, the antibody is humanized. Asused herein, “humanized” describes antibodies wherein some, most or allof the amino acids outside the CDR regions are replaced withcorresponding amino acids derived from human immunoglobulin molecules.In one embodiment of the humanized forms of the antibodies, some, mostor all of the amino acids outside the CDR regions have been replacedwith amino acids from human immunoglobulin molecules but where some,most or all amino acids within one or more CDR regions are unchanged.Small additions, deletions, insertions, substitutions or modificationsof amino acids are permissible as long as they would not abrogate theability of the antibody to bind a given antigen. Suitable humanimmunoglobulin molecules would include IgG1, IgG2, IgG3, IgG4, IgA andIgM molecules. A “humanized” antibody would retain a similar antigenicspecificity as the original antibody, i.e., in the present invention,the ability to bind CCR5.

One skilled in the art would know how to make the humanized antibodiesof the subject invention. Various publications, several of which arehereby incorporated by reference into this application, also describehow to make humanized antibodies. For example, the methods described inU.S. Pat. No. 4,816,567 (45) comprise the production of chimericantibodies having a variable region of one antibody and a constantregion of another antibody.

U.S. Pat. No. 5,225,539 (46) describes another approach for theproduction of a humanized antibody. This patent describes the use ofrecombinant DNA technology to produce a humanized antibody wherein theCDRs of a variable region of one immunoglobulin are replaced with theCDRs from an immunoglobulin with a different specificity such that thehumanized antibody would recognize the desired target but would not berecognized in a significant way by the human subject's immune system.Specifically, site directed mutagenesis is used to graft the CDRs ontothe framework.

Other approaches for humanizing an antibody are described in U.S. Pat.Nos. 5,585,089 (47) and 5,693,761 (48) and WO 90/07861 which describemethods for producing humanized immunoglobulins. These have one or moreCDRs and possible additional amino acids from a donor immunoglobulin anda framework region from an accepting human immunoglobulin. These patentsdescribe a method to increase the affinity of an antibody for thedesired antigen. Some amino acids in the framework are chosen to be thesame as the amino acids at those positions in the donor rather than inthe acceptor. Specifically, these patents describe the preparation of ahumanized antibody that binds to a receptor by combining the CDRs of amouse monoclonal antibody with human immunoglobulin framework andconstant regions. Human framework regions can be chosen to maximizehomology with the mouse sequence. A computer model can be used toidentify amino acids in the framework region which are likely tointeract with the CDRs or the specific antigen and then mouse aminoacids can be used at these positions to create the humanized antibody.

The above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 (49)also propose four possible criteria which may used in designing thehumanized antibodies. The first proposal was that for an acceptor, use aframework from a particular human immunoglobulin that is unusuallyhomologous to the donor immunoglobulin to be humanized, or use aconsensus framework from many human antibodies. The second proposal wasthat if an amino acid in the framework of the human immunoglobulin isunusual and the donor amino acid at that position is typical for humansequences, then the donor amino acid rather than the acceptor may beselected. The third proposal was that in the positions immediatelyadjacent to the 3 CDRs in the humanized immunoglobulin chain, the donoramino acid rather than the acceptor amino acid may be selected. Thefourth proposal was to use the donor amino acid reside at the frameworkpositions at which the amino acid is predicted to have a side chain atomwithin 3 Å of the CDRs in a three dimensional model of the antibody andis predicted to be capable of interacting with the CDRs. The abovemethods are merely illustrative of some of the methods that one skilledin the art could employ to make humanized antibodies.

In one embodiment, the antibody of the subject invention is detectable.In one embodiment of the above antibody, the detectable antibody islabeled with a detectable marker as described above.

This invention provides a composition comprising the above antibody andan agent conjugated to the antibody. In one embodiment, the agent is aradioactive isotope or toxin.

This invention provides a method of determining whether a subject isafflicted with a disease associated with Kaposi's sarcoma-associatedherpesvirus (KSHV) infection of a B cell which comprises: (a) obtaininga suitable sample from the subject; (b) contacting the suitable samplewith a detectable antibody capable of binding to Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide or a fragment thereof so as to form a complex between theantibody and any Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 polypeptide or a fragment thereofpresent in the sample; (c) removing any unbound antibody; and (d)detecting any antibody which is bound to any Kaposi's sarcoma-associatedherpesvirus latency-associated nuclear antigen 2 polypeptide or afragment thereof in the sample, wherein the presence of antibodyindicates that the subject is afflicted with the disease associated withKaposi's sarcoma-associated herpesvirus infection of a B cell.

This invention provides a method of determining whether a subject isafflicted with a disease associated with Kaposi's sarcoma-associatedherpesvirus infection of a B cell which comprises: (a) obtaining asuitable sample from the subject; (b) immobilizing a capturing antibodywherein the capturing antibody is capable of binding to Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2 to asupport; (c) removing any unbound capturing antibody; (d) contacting thecapturing antibody with the suitable sample so as to form a complexbetween the antibody and any Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 present in the sample; (e) removingany unbound sample; (f) contacting the complex obtained in step (d) witha detectable antibody of Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 polypeptide or a fragment thereofso as to form a complex between the detectable antibody and the complex;(g) removing any unbound detectable antibody; and (h) detecting anydetectable antibody which is bound to the complex wherein the presenceof detectable antibody indicates that the subject is afflicted with thedisease associated with Kaposi's sarcoma-associated herpesvirusinfection of a B cell.

The disease in the above methods includes but is not limited toCastleman's disease and Primary Effusion Lymphoma. The disease may alsobe one not associated with a B cell.

This invention provides a method of determining whether a subject isinfected with Kaposi's sarcoma-associated herpesvirus which comprises:(a) obtaining a suitable sample from the subject; (b) contacting thesuitable sample with the detectable antibody of Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide or a fragment thereof so as to form a complex between theantibody and any polypeptide or fragment thereof present in the sample;(c) removing any unbound antibody; and (d) detecting any antibody whichis bound to any Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 polypeptide or fragment thereof inthe sample, wherein the presence of antibody indicates that the subjectis infected with Kaposi's sarcoma-associated herpesvirus.

This invention provides a method of determining whether a subject isinfected with Kaposi's sarcoma-associated herpesvirus which comprises:(a) obtaining a suitable sample from the subject; (b) immobilizing acapturing antibody wherein the capturing antibody is capable of bindingto polypeptide or fragment thereof to a support; (c) removing anyunbound capturing antibody; (d) contacting the capturing antibody withthe suitable sample so as to form a complex between the antibody andKaposi's sarcoma-associated herpesvirus latency-associated nuclearantigen 2 polypeptide or fragment thereof present in the sample; (e)removing any unbound sample; (f) contacting the complex obtained in step(d) with the detectable antibody which is bound to Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide or fragment thereof so as to form a complex between thedetectable antibody and the complex; (g) removing any unbound detectableantibody; and (h) detecting any detectable antibody which is bound tothe complex wherein the presence of detectable antibody indicates thatthe subject is infected with Kaposi's sarcoma-associated herpesvirus.

The suitable sample includes but is not limited to tonsil tissue, lymphnodes, spleen, skin lesions, blood, serum, plasma, cerebrospinal fluid,lymphocytes, urine, transudates, exudates, bone marrow cells, orsupernatant from a cell culture.

In one embodiment of the above method, the antigen bound by the antibodyis detected by an immunoassay. The immunoassay of the above methodincludes but is not limited to ELISA, IFA, and Western blotting.

As used herein, “capturing antibody” refers to an antibody capable ofbinding a polypeptide, a second antibody or a complex comprising anantibody and a polypeptide as described above. In one embodiment, acapturing antibody binds to a different epitope on the target proteinthan the detecting antibody.

As used herein, “support” includes but is not limited to a solidsurface, a bead, a column, a plastic dish, a plastic plate, a microscopeslide, and a nylon membrane. The use of these and other supports areknown by one skilled in the art.

This invention provides a kit for diagnosing Kaposi's sarcoma-associatedherpesvirus infection comprising the labeled antibody capable ofspecifically binding to the Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 polypeptide or fragment thereof. Inone embodiment of the above kit, the kit further comprises a means fordetermining the level of Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 polypeptide or fragment thereofbound by an antibody. In one embodiment of the above kit, the labeledantibody capable of specifically binding to the polypeptide encoded bythe isolated nucleic acid of Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 polypeptide or fragment thereof isbound to a support.

Studies have shown that LANA2 polypeptide can inhibit p53 mediatedapoptosis. Accordingly, this invention provides a method of inhibitingp53 mediated apoptosis of a cell which comprises introducing into thecell an effective amount of the replicable vector which comprises theisolated nucleic acid which encodes Kaposi's sarcoma-associatedherpesvirus latency-associated nuclear antigen 2 polypeptide or fragmentthereof, so as to thereby inhibit p53 mediated apostosis of the cell.

This invention provides a method of immortalizing a cell which comprisesintroducing into the cell an amount of the replicable vector whichcomprises the isolated nucleic acid which encodes Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide or fragment thereof effective to inhibit p53 mediatedapoptosis of the cell, so as to thereby immortalize the cell.

As used herein, “immortalizing” refers to the action of LANA2polypeptide in a B cell wherein the LANA2 polypeptide interacts with thep53 mediated apoptosis pathway to inhibit the action of p53 in the cell.The above interaction does not allow the cell to die, thereby creatingan “immortalized” cell.

This invention provides a method of producing an antibody whichcomprises introducing into a cell an amount of the replicable vectorwhich comprises the isolated nucleic acid which encodes Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide effective to inhibit p53 mediated apoptosis of the cellproducing the antibody and thereby immortalizing the cell, so as to isthereby produce the antibody. An application of this method is toimmortalize a cell which produces an antibody so to thereby increaseproduction of the antibody.

The cell in the above methods includes but is not limited to ahematopoietic tissue cell, and a B cell.

As used herein, the term “introducing into a cell” includes but is notlimited to transduction and transfection. Transfection can be achievedby calcium phosphate co-precipitates, conventional mechanical proceduressuch as micro-injection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors or any other method known to one skilledin the art. This invention provides an antibody produced by the abovemethod.

This invention provides a method of determining whether a subject isinfected with Kaposi's sarcoma-associated herpesvirus which comprises:(a) obtaining a suitable sample from the subject; (b) contacting thesuitable sample with a detectable nucleic acid capable of hybridizing toa nucleic acid which encodes Kaposi's sarcoma-associated herpesviruslatency-associated nuclear antigen 2 polypeptide or fragment thereofunder hybridizing conditions so as to form a complex between thedetectable nucleic acid and any nucleic acid which encodes a Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide or fragment thereof which is present in the sample; (c)removing any unbound detectable nucleic acid; and (d) detecting anydetectable nucleic acid which is bound to the complex, wherein thepresence of detectable nucleic acid indicates that the subject isinfected with Kaposi's sarcoma-associated herpesvirus.

In one embodiment of the above method, the suitable sample includes butis not limited to tonsil tissue, lymph nodes, spleen, skin lesions,blood, serum, plasma, cerebrospinal fluid, lymphocytes, urine,transudates, exudates, bone marrow cells, or supernatant from a cellculture.

In one embodiment of the above methods, the subject is a mouse, rat,dog, guinea pig, ferret, rabbit, primate, and human. As used herein,“subject” means any animal or artificially modified animal capable ofbecoming KSHV infected. Artificially modified animals include, but arenot limited to, SCID mice with human immune systems. The subjectsinclude but are not limited to mice, rats, dogs, guinea pigs, ferrets,rabbits, and primates. In the preferred embodiment, the subject is ahuman being.

This invention provides a kit for diagnosing Kaposi's sarcoma-associatedherpesvirus infection comprising a labeled nucleic acid which encodes aKaposi's sarcoma-associated herpesvirus latency-associated nuclearantigen 2 polypeptide or fragment thereof. In one embodiment of theabove kit, the kit further comprises a means for determining the levelof sample bound to the above labeled nucleic acid. In one embodiment ofthe above kit, the above labeled nucleic acid is bound to a support.

This invention provides a transgenic non-human animal which has stablyintegrated into the genome of its germ cells or somatic cells anexogenous nucleic acid construct wherein the nucleic acid constructcomprises a B-cell specific promoter of Kaposi's sarcoma-associatedherpesvirus latency-associated nuclear antigen 2 operably linked to asecond nucleic acid which encodes a gene of interest and is introducedinto the transgenic non-human animal, or an ancestor, at an embryonicstage. In one embodiment of the above transgenic animal, the animal is amammal. In one embodiment of the above transgenic animal, the non-humananimal is a mouse, a rat, a sheep, a dog, a primate, or a reptile.

This invention provides a method for evaluating in a non-humantransgenic animal the potential therapeutic effect of an agent fortreating Kaposi's sarcoma-associated herpesvirus infection in a human,which comprises: (a) providing an agent to a transgenic non-human animalwhose cells comprise the nucleic acid which encodes a Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide; and (b) determining the therapeutic effect of the agent onthe transgenic non-human animal by monitoring Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2expression, wherein a decrease in Kaposi's sarcoma-associatedherpesvirus latency-associated nuclear antigen 2 indicates that theagent would have a potential therapeutic effect on Kaposi'ssarcoma-associated herpesvirus infection in a human. In one embodimentof the above method, the animal is a mammal. In one embodiment of theabove method, the non-human animal is a mouse, a rat, a sheep, a dog, aprimate, or a reptile.

The following U.S. patents are hereby incorporated by reference: U.S.Pat. No. 6,025,539, IL-S transgenic mouse; U.S. Pat. No. 6,023,010,Transgenic non-human animals depleted in a mature lymphocytic cell-type;U.S. Pat. No. 6,018,098, In vivo and in vitro model of cutaneousphotoaging; U.S. Pat. No. 6,018,097, Transgenic mice expressing humaninsulin; U.S. Pat. No. 6,008,434, Growth differentiation factor-litransgenic mice; U.S. Pat. No. 6,002,066; H2-M modified transgenic mice;U.S. Pat. No. 5,994,618, Growth differentiation factor-8 transgenicmice; U.S. Pat. No. 5,986,171, Method for examining neurovirulence ofpolio virus, U.S. Pat. No. 5,981,830, Knockout mice and their progenywith a disrupted hepsin gene; U.S. Pat. No. 5,981,829, .DELTA.Nur77transgenic mouse; U.S. Pat. No. 5,936,138; Gene encoding mutant L3T4protein which facilitates HIV infection and transgenic mouse expressingsuch protein; U.S. Pat. No. 5,912,411, Mice transgenic for atetracycline-inducible transcriptional activator; U.S. Pat. No.5,894,078, Transgenic mouse expressing C-100 app.

The methods used for generating transgenic animals are well known to oneof skill in the art. For example, one may use the manual entitled“Manipulating the Mouse Embryo” by Brigid Hogan et al. (Ed. Cold SpringHarbor Laboratory) 1986.

See for example, Leder and Stewart, U.S. Pat. No. 4,736,866 for methodsfor the production of a transgenic mouse.

For sometime it has been known that it is possible to carry out thegenetic transformation of a zygote (and the embryo and mature organismwhich result therefrom) by the placing or insertion of exogenous geneticmaterial into the nucleus of the zygote or to any nucleic geneticmaterial which ultimately forms a part of the nucleus of the zygote. Thegenotype of the zygote and the organism which results from a zygote willinclude the genotype of the exogenous genetic material. Additionally,the inclusion of exogenous genetic material in the zygote will result ina phenotype expression of the exogenous genetic material.

The genotype of the exogenous genetic material is expressed upon thecellular division of the zygote. However, the phenotype expression,e.g., the production of a protein product or products of the exogenousgenetic material, or alterations of the zygote's or organism's naturalphenotype, will occur at that point of the zygote's or organism'sdevelopment during which the particular exogenous genetic material isactive. Alterations of the expression of the phenotype include anenhancement or diminution in the expression of a phenotype or analteration in the promotion and/or control of a phenotype, including theaddition of a new promoter and/or controller or supplementation of anexisting promoter and/or controller of the phenotype.

The genetic transformation of various types of organisms is disclosedand described in detail in U.S. Pat. No. 4,873,191, issued Oct. 10,1989, which is incorporated herein by reference to disclose methods ofproducing transgenic organisms. The genetic transformation of organismscan be used as an in vivo analysis of gene expression duringdifferentiation and in the elimination or diminution of genetic diseasesby either gene therapy orby using a transgenic non-human mammal as amodel system of a human disease. This model system can be used to testputative drugs for their potential therapeutic value in humans.

The exogenous genetic material can be placed in the nucleus of a matureegg. It is preferred that the egg be in a fertilized or activated (byparthenogenesis) state. After the addition of the exogenous geneticmaterial, a complementary haploid set of chromosomes (e.g., a sperm cellor polar body) is added to enable the formation of a zygote. The zygoteis allowed to develop into an organism such as by implanting it in apseudopregnant female. The resulting organism is analyzed for theintegration of the exogenous genetic material. If positive integrationis determined, the organism can be used for the in vivo analysis of thegene expression, which expression is believed to be related to aparticular genetic disease.

The “transgenic non-human animals” of the invention may be produced byintroducing “transgenes” into the germline of the non-human animal.Embryonal target cells at various developmental stages can be used tointroduce transgenes. Different methods are used depending on the stageof development of the embryonal target cell. The zygote is the besttarget for micro-injection. In the mouse, the male pronucleus reachesthe size of approximately 20 micrometers in diameter which allowsreproducible injection of 1-2 pl of DNA solution. The use of zygotes asa target for gene transfer has a major advantage in that in most casesthe injected DNA will be incorporated into the host gene before thefirst cleavage (Brinster, et al. (1985) Proc. Natl. Acad. Sci. U.S.A.82, 4438-4442). As a consequence, all cells of the transgenic non-humananimal will carry the incorporated transgene. This will in general alsobe reflected in the efficient transmission of the transgene to offspringof the founder since 50% of the germ cells will harbor the transgene.Microinjection of zygotes is the preferred method for incorporatingtransgenes in practicing the invention.

Retroviral infection can also be used to introduce transgene into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, R. (1976) Proc. Natl. Acad.Sci U.S.A. 73, 1260-1264). Efficient infection of the blastomeres isobtained by enzymatic treatment to remove the zona pellucida (Hogan, etal. (1986) in Manipulating the Mouse Embryo, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) The viral vector system usedto introduce the transgene is typically a replication-defectiveretrovirus carrying the transgene (Jahner, et al. (1985) Proc. Natl.Acad. Sci. U.S.A. 82, 6927-6931; Van der Putten, et al. (1985) Proc.Natl. Acad. Sci U.S.A. 82, 6148-6152). Transfection is easily andefficiently obtained by culturing the blastomeres on a monolayer ofvirus-producing cells (van der Putten, supra; Stewart, et al. (1987)EMBO J. 6, 383-388). Alternatively, infection can be performed at alater stage. Virus or virus-producing cells can be injected into theblastocoele (Jahner, D., et al. (1982) Nature 298, 623-628) Most of thefounders will be mosaic for the transgene since incorporation occursonly in a subset of the cells which formed the transgenic non-humananimal. Further, the founder may contain various retroviral insertionsof the transgene at different positions in the genome which generallywill segregate in the offspring. In addition, it is also possible tointroduce transgenes into the germ line, albeit with low efficiency, byintrauterine retroviral infection of the midgestation embryo (Jahner, D.et al. (1982) supra).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans, M. J., et al. (1981)Nature 292, 154-156; Bradley, M. O., et al. (1984) Nature 309, 255-258;Gossler, et al. (1986) Proc. Natl. Acad. Sci U.S.A. 83, 9065-9069; andRobertson, et al. (1986) Nature 322, 445-448). Transgenes can beefficiently introduced into the ES cells by DNA transfection or byretrovirus-mediated transduction. Such transformed ES cells canthereafter be combined with blastocysts from a non-human animal. The EScells thereafter colonize the embryo and contribute to the germ line ofthe resulting chimeric animal. For review see Jaenisch, R. (1988)Science 240, 1468-1474.

As used herein, a “transgene” is a DNA sequence introduced into thegermline of a non-human animal by way of human intervention such as byway of the above described methods.

This invention provides a method of treating Kaposi's sarcoma-associatedherpesvirus infection in a subject, which comprises introducing into thesubject's cells an effective amount of the nucleic acid capable ofspecifically hybridizing to the isolated nucleic acid which encodesKaposi's sarcoma-associated latency-associated nuclear antigen 2polypeptide or fragment thereof to hybridize to any of the above nucleicacid which is present in the subject's cells, so as to thereby treatKaposi's sarcoma-associated herpesvirus infection. An application ofthis method is to inhibit transcription of Kaposi's sarcoma-associatedlatency-associated nuclear antigen 2 polypeptide or fragment thereof,thereby treating the subject.

As used herein, “effective amount” means an amount in sufficientquantities to either treat the subject or prevent the subject frombecoming infected with Kaposi's sarcoma-associated herpesvirus. A personof ordinary skill in the art can perform simple titration experiments todetermine what amount is required to treat the subject.

The subject invention has various applications which includes KSHVtreatment such as treating a subject who has become afflicted with KSHV.As used herein, “afflicted with the disease” means that the subject hasat least one cell which has been infected by KSHV. As used herein,“treating” means either slowing, stopping or reversing the progressionof an HIV-1 disorder. In the preferred embodiment, “treating” meansreversing the progression to the point of eliminating the disorder. Asused herein, “treating” also means the reduction of the number of viralinfections, reduction of the number of infectious viral particles,reduction of the number of virally infected cells, or the ameliorationof symptoms associated with KSHV.

Another application of the subject invention is to prevent a subjectfrom contracting KSHV. As used herein, “contracting KSHV” means becominginfected with KSHV, whose genetic information replicates in and/orincorporates into the host cells. Another application of the subjectinvention is to treat a subject who has become infected with KSHV.

As used herein, “KSHV infection” means the introduction of KSHV geneticinformation into a target cell, such as by fusion of the target cellmembrane with KSHV or an KSHV envelope glycoprotein cell. The targetcell may be a bodily cell of a subject. In the preferred embodiment, thetarget cell is a bodily cell from a human subject.

This invention provides a method of treating Kaposi's sarcoma-associatedherpesvirus infection in a subject, which comprises introducing into thesubject's cells an effective amount of a nucleic acid capable ofspecifically hybridizing to an isolated nucleic acid comprisingnucleotides having the sequence of a promoter of latency-associatednuclear antigen 2 transcription to hybridize to any of this nucleic acidwhich is present in the subject's cells, so as to thereby treat thesubject. An application of this method is to hybridize a nucleic acid tothe above promoter, thereby inhibiting Kaposi's sarcoma-associatedherpesvirus latency-associated nuclear antigen 2 polypeptide expression,thereby treating Kaposi's sarcoma-associated herpesvirus infection inthe subject.

This invention provides a composition comprising the antibody capable ofspecifically binding to the polypeptide encoded by the isolated nucleicacid which encodes Kaposi's sarcoma-associated latency-associatednuclear antigen 2 polypeptide and a carrier.

As used herein, “carriers” include but are not limited to aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, saline and buffered media.Parenteral vehicles include sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, chelating agents, inertgases and the like. The carriers include but are not limited to anaerosol, intravenous, oral or topical carrier. Carriers are well knownto those skilled in the art.

As used herein, “composition” means a mixture. The compositions includebut are not limited to those suitable for oral, rectal, intravaginal,topical, nasal, opthalmic, or parenteral administration to a subject. Asused herein, “parenteral” includes but is not limited to subcutaneous,intravenous, intramuscular, or intrasternal injections or infusiontechniques.

This invention provides a method of treating a subject infected withKaposi's sarcoma-associated herpesvirus, which comprises administeringto the subject an amount of the above composition under conditions suchthat the antibody binds to any LANA2 present in the subject, so as tothereby treat the subject.

This invention provides a composition comprising the polypeptide encodedby the isolated nucleic acid which encodes Kaposi's sarcoma-associatedlatency-associated nuclear antigen 2 polypeptide or fragment thereof anda carrier.

In one embodiment of the methods of this invention, the cell is presentin a subject and the contacting is effected by administering thecompound to the subject.

The subject invention has therapeutic applications. For example, oneskilled in the art can target a latency associated gene, such as thegene which encodes the LANA2 polypeptide, so as to inactivate the geneand thereby treat the KSHV infection or other diseases associated withKSHV infection, such as the B-cell associated diseases Castleman'sdisease or Primary Effusion Lymphoma. One can use antisense technologyin order to inhibit the expression of a gene, such as LANA2. One canalso use monoclonal antibody technology so as to degrade or sequesterthe protein, such as the LANA2 protein.

This invention will be better understood from the Experimental Detailsthat follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims that followthereafter.

EXPERIMENTAL DETAILS

A. Materials and Methods

Cell Cultures

BC-1, BCP-1, BCBL-1, BJAB, Ramos, and P3HR1 (obtained from the AmericanType Culture Collection—ATCC) cells were maintained in 1640 RPMI(GibcoBRL, Gaithersburg, Md.) supplemented with 10-20% fetal bovineserum (GibcoBRL). SAOS-2, U2OS, COS7 cells (obtained from ATCC), andIRF1/2 (−/−) cells (a gift from T. Taniguchi (43)) were maintained inDMEM (GibcoBRL) with 10% fetal bovine serum. Induction of viral lyticreplication and gene transcription was performed by treatment of cellswith 20 ng/ml TPA (Sigma Chemical Co., St. Louis, Mo.). Cells wereharvested 48 h after treatment. To inhibit viral DNA replication, PFA(Sigma) was added at a concentration of 0.5 mM either or in the presenceof 20 mg/ml TPA for 48 h.

Northern Analysis

Total RNA was extracted by the RNAzol method (TelTest, Friendswood,Tex.) followed by mRNA selection using a PolyATract mRNA isolation kit(Promega, Madison, Wis.). Five hundred nanograms of the poly(A)-selectedmRNA was loaded per lane on formaldehyde 1% agarose gel and transferredonto nylon membranes (GeneScreen, NEN Research Products, Boston, Mass.).The V1 probe consists of the entire K9 ORF (14). The V1 probe as well asV2, V3 and V4 probes derived from PCR products (see FIG. 2)

-   (V2F: 5′-GGGAATTCGATGCCTAAAGCCGGTGGC-31 and-   V2R: 5′-TGCGGCCGCTCAAACCTCACACCCCCT-3′;-   V3F: 5′-GGGAATTCGATGTACCACGTGGGACAG-3′ and-   V3R: 5′-TGCGGCCGCTTAGTCATCACATGTAAC-3′;-   V4F: 5′-GGGAATTCGATGCCTCGCTACACGGAG-3′ and-   V4R: 5′-GGGAATTCGCTACCTCTGGGCTTTTTT-3′) were labeled by random    priming using synthetic hexanucleotide primers (RediPrime, DNA    labeling system; Amersham International, Amersham, England) and    [³⁵P]dCTP. Hybridization was performed in 5×SSC (1×SSC is 0.15 M    NaCl plus 0.015 M sodium citrate)-50% formamide-5× Denhardt's    solution-2% sodium dodecyl sulfate-10% dextran sulfate-100 mg of    denatured sheared salmon sperm DNA per ml at 42° C. b-actin probe    was used to standardize the amount of RNA loaded.    cDNA Library Screening

cDNA phage libraries of TPA stimulated BC-1 cells were constructed andamplified in the ZAP Express™ vector according to the manufacturer'sprotocol (Stratagene, La Jolla, Calif.). 3.2×10′ plaques were, screenedfollowing manufacturer's suggested protocols with the V3 probe made fromthe V3F and V3R PCR primers (used in northern analysis, see above).

Plasmids

pcDNA.LANA2 was obtained by excising full length LANA2 insert with EcoRIand NotI digestion from phagemid, pBK-CMV-LANA2 f703 screened from cDNAlibrary). This insert was then cloned in frame into EcoRI/NotI preparedpcDNAHis3.1B vector (Invitrogen, Carlsbad, Calif.). pMET7.LANA2 wasconstructed by digesting pBK-CMV-LANA2 with PstI and XbaI.

The isolated insert was then cloned into PstI/XbaI prepared pMET7mammalian expression vector (41). The fidelity of all cloning junctionswas verified on an ABI 377 Sequenator (Applied Bio-systems Inc., FosterCity, Calif.) pG13-Luc, a reporter plasmid containing 13 tandemp53-response elements derived from the p21 promoter, was a gift from W.El-Deiry and B. Volgelstein (4). pGL-3 control (Promega, Madison, Wis.)was used as a control vector for luciferase transient transfectionassays. GST-p53 (full length (FL)) and the C-terminus fragment of p53(GST-p53 (290-393)) plasmids were a gift from W. Gu (17). DNA sequencescorresponding to the 1-100 and 100-290 aminoacids of human p53 wereamplified by PCR and subcloned into pGEX-KG (18) to generate the proteinexpression plasmids GST-p53 (1-100) and GST-p53 (100-290). pcDNA.p53expression plasmid was a gift of RT Hay (35). pEGFP—F* (gift of W.Jiang) expresses green fluorescent protein (GFP) and was used as amarker for pcDNA.LANA2 and/or pcDNA.p53 transfection to gate fluorescentcells by FACS. The plasmid containing the Gal-4 binding domain(Gal4-BD), PAS2-1, the Gal4-activation domain (Gal4-AD), pGAD424, aswell as the plasmids containing the DNA-BD/murine p53 fusion proteinPVA3 and the DNA-AD/murine p53 fusion protein pGADp53 and controlplasmids pCL1, PLAM5′, pGBT9 and PTD1 were obtained from Clontech(Clontech laboratories, Palo Alto, Calif.).

Reporter Assays

SAOS-2 or U2OS cells were seeded at a density of 5×10⁴ cells per platein six-well plates 1 day before transfection. Transient transfectionswith plasmid DNA were performed using Cell Phect (Pharmacia Biotech,Piscataway, N.J.). In all experiments, total amounts of transfected DNAwere equalized between wells using empty pcDNA3.1H is C (Invitrogen).Cells were harvested and lysed, and luciferase activity was measured byusing standard protocols after 48 hr. pcDNAHis3.1LacZ (Invitrogen) wasused to normalize luciferase activity to transfection efficiency. Inthis way, reporter expression levels were normalized to the amount oftransfected plasmid for each experimental condition. Each measurementwas performed in triplicate, with experiments independently replicatedat least three times. p53-null SAOS-2 cells were co-transfected with 2mg pG13-Luc in the presence or absence of 0.5 mg pcDNA.p53 with orwithout pcDNA.LANA2 (0.5-1 mg). U2OS cells were co-transfected with 2 mgpG13-Luc in the presence or absence of pcDNA.LANA2 (0.5-1 mg/well) andtreated with 0.4 mM doxorubicin (Sigma) 18 h post-transfection.

Fluorescence Activated Cell Sorter (FACS) Analysis

1×10⁶ SAOS-2 cells were transfected (Cell Phect) with 1 mg of the GFPexpressing plasmid, pEGFP—F*, in the presence of pcDNA.p53 (4.5 mg)and/or pcDNA.LANA2 (4.5 mg) or the empty expression vector. U2OS cellswere transfected with 1 mg pEGFP—F* in the presence or absence of theexpression vector pcDNA.LANA2 (4.5-9 mg) and treated with doxorubicin 18h post-transfection. 48 h after transfection, cells were washed in PBSand fixed at 4° C. in 80% ethanol in PBS for 1 h. Cells were then washedthree times with PBS and incubated for 30 min at 37° C. in 0.1% TritonX-100, 0.1% Trisodium citrate, 0.5 mg/ml RNaseA, and 50 mg/ml propidiumiodide. The DNA content of cells gated for GFP expression was thenanalyzed using a FACScan flow cytometer.

Activation of Caspase-8

Caspase-8 activation was determined using the synthetic oligopeptidesubstrate Ac-LETD-AFC from Bio-Rad (Bio-Rad Laboratories, Hercules,Calif.), as described by the manufacturer and the samples were read on aBio-Rad VersaFluor™ Fluorometer.

GST Pull Down Assays

GST in vitro binding assays were performed using in vitro translated[³⁵S]methionine-labeled LANA2 incubated with p53 GST fusion proteins(GST-p53 (FL), GST-p53 (1-100), GST-p 53 (100-290), GST-p53 (290-393),and GST alone. In vitro translated [S³⁵]methionine-labeled p53 wasincubated with GST-LANA2 and GST alone.

Coimmunoprecipitation

LANA2 (20 mg of pcDNA.LANA2) and p53 (20 mg of pcDNA.p53) were expressedin SAOS-2 cells by co-transfection and were immunoprecipitated withanti-LANA2 CM-8B6 or CM-10A2 antibodies, or DO-1 (Santa Cruz Biotech,Santa Cruz, Calif.), Pab 1801 (Santa Cruz,), and Ab-1 (Oncogene,Cambridge, Mass.) anti-p53 antibodies. Protein complexes were resolvedby SDS/10% PAGE and transferred onto nitrocellulose membrane. LANA2 wasdetected using CM-8B6, CM-10A2 and p53 was detected using DO-1, Pab1801, Ab-1 by immunoblotting and enhanced chemiluminescence (ECL,Amersham, Piscataway, N.J.).

Immunohistochemistry of KSHV Infected Tissues and Controls

Glass slides were obtained with the Cytospin 3 apparatus (ShandonLipshaw, Pittsburgh, Pa.) using 25,000 washed cells per spot. Thesecytospins were air-dried overnight, fixed in acetone for 4 minutes atroom temperature, air-dried for 30 minutes and processed forimmunohistochemistry. Ten KS skin lesions, 5 lymph nodes from patientswith CD, and biopsies from two cases of PEL were investigated forprotein expression of LANA2 by immunohistochemistry. One CD lymph nodealso contained KS. Control tissues were tonsil biopsies from KSHVnegative children. Mouse monoclonal antibody, clone CM-10A2, was madeagainst bacterially produced GST-LANA2 and was confirmed to be specificto the 80 kD LANA2 protein on western blot hybridization ofKSHV-infected cell lysates (BC-1, BCBL-1, BCP-1) compared toKSHV-uninfected cell lysates (BJAB, P3HR1, Ramos). CM-10A2 wasnon-reactive to GST protein by both ELISA and western blothybridization. The rabbit polyclonal antibody against LANA1, R UK163,was the kind gift of B. Chandran. Microwave-ethylenediaminetetraaceticacid (EDTA) pretreatment was required for antigen retrieval. Antibodybinding was revealed using peroxidase-labeled goat anti-mouse antisera(DAKO, Glostrupp, Denmark) followed by tyramide amplification(DuPont/NEN, Boston, Mass.). Reactions were developed usingdiaminobenzidine (DAB; Sigma) or amino ethyl carbazole (AEC; DAKO) aschromogenic substrates, and sections were counterstained withhematoxylin. Antibodies to KSHV vIL-6 (cytoplasmic staining) and PF-8(perinuclear staining) were used for comparative antibody controls fortissue staining (32). For fluorescence double-immunostaining with LANA1and LANA2, fluorescein-isothiocyanate (FITC)-conjugated goat anti-mousewas used in combination with goat anti-rabbit antisera (SouthernBiotechnology) followed by Avidin Texas Red (Vector Laboratories,Burlingame, Calif.).

Serologic Analysis

COS7 cells were plated at a density of 10⁶ cells per 90 mm plate one dayprior to transfection. Transfections were performed (Cell Phect) using 6mg of pMET7.LANA2 or pMET7 empty vector as control. Cells were harvested48 hr post-transfection, placed in 500 ml lysis buffer, incubated on icefor 20 minutes, centrifuged, and resuspended in 200 ul nuclearextraction buffer (20 mM HEPES pH7.9, 0.4M NaCl, 1 mM EDTA, 1 mM DTT, 1mM PMSF, 1 mM sodium vanadate, lug/ml aprotinin, leupeptin andpepstatin). 15 ug of protein was loaded into a single well comb forwestern blot analysis by SDS/12.5% PAGE. After transferring ontonitrocellulose membrane, strips were cut and incubated in CM-10A2primary antibody overnight. After washing, strips were incubated for onehour in anti-human IgG alkaline phosphatase conjugate secondary antibody(1:3500 dilution; Sigma).

Yeast Two-Hybrid Assay

LANA2 was fused either to GAL4-AD in the plasmid pGAD424 or to GAL4DNA-binding domain (BD) in the plasmid pAS2-1.

The plasmids containing the murine p53 fused to GAL4 AD or GAL4BD wereprovided by Clontech. The yeast strain Y-190 was used for this twohybrid assay. Plasmids are introduced into Y-190 by the standard lithiumacetate transformation method.

To test for potential protein—protein interaction, transformants werescreened for growth in medium lacking histidine but in the presence of15 mM 3-aminotriazol (3-AT) (His+phenotype) or assayed forb-galactosidase activity (blue phenotype) in the presence of X-Gal(5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside).

B. Results

Identification of the K10.5 (LANA2) Transcript

ORF K10.5 was originally described as a sequence feature rather than anopen reading frame in Russo et al.'s conservative annotation of the BC-igenome (36). This gene was one of four KSHV sequences showing limitedhomology to cellular IRFs. Neipel and colleagues subsequently annotateda theoretical ORF K10.1 based on their sequencing of the KSHV genomefrom a KS lesion (30). We therefore sought to directly determine whetherthe four IRF-like motifs, including the putative K10.1 gene, in the KSHVgenomic sequence represent expressed gene products using TPA-induced anduninduced BC-1 cell mRNAs.

Four probes spanning nt 83,860-85,209 (V1 probe), nt 88,409-88,909 (V2probe), nt 89,599-90,540 (v3 probe) and nt. 93,635-94,126 (V4 probe)were generated by PCR (see FIG. 2). V1 probe corresponds to the ORFK9region-encoding VIRF and expression patterns for this probe matchedthose previously described (27, 29, 37) in that the 1.5 kb mRNA isweakly detected in unstimulated BC-1 cells and induced to high levels ofexpression after TPA treatment. While probes V2 and V4 (corresponding tothe vIRF2 protein gene (6)) did not hybridize to detectable transcriptsin unstimulated or stimulated BC-1 cells, the V3 probe corresponding toORFK10.5 hybridized to a 1.8 kb transcript that was absent from the KSHVnegative control cell line, P3HR1 (FIG. 1). Expression of the K10.5transcript is not affected by TPA stimulation or phosphonoformic acid(PFA) inhibition thereby qualifying it as a latent transcript in BC-1cells. Similar results were also obtained using BCBL-1 cells (data notshown).

Since the transcript size identified by the V3 probe is incompatiblewith the predicted transcript for putative ORFK10.5, we screened a cDNAlibrary made from TPA-stimulated BC-1 cells to identify splicedtranscripts. Of 3.2×10′ plaques screened, six positive phages were foundwith inserts ranging between 503 bp and 1735 bp in length (FIG. 2). Theclones were sequenced and one (f703) contained the full length cDNAtranscript beginning 31 bp upstream from a putative start ATG (nt91,393) and having a stop codon at nt 89,599. All six cDNAs have a 3′termination coordinate at nt 89,599. Conserved splice-donor sites (nt90,938 and nt 90,847) are present in the f703 insert, but only one ofthe five other phage inserts extended through the 5′ splice junction.Splicing results in a 1704 bp full length transcript for the newlyannotated gene which is designated ORFK10.5 to distinguish it from theunspliced 3′ exon previously designated K10.1 (FIG. 2, GenBank AccessionNo. A4008303). This ORF is composed of a novel 455 bp 5′ exon that isjoined to the 3′ exon 1339 bp internally to and out of frame with thepreviously annotated ORF K10.1 predicted from the genome sequenceanalysis (30).

Based on its constitutive expression in BC-1 cells and its nuclearlocalization (see below), we refer to the protein encoded by ORFK10.5 aslatency-associated nuclear antigen 2 (LANA2). LANA2 has low overallhomology to members of the IRF family. Members of the IRF family ofproteins have at least two common functional domains: an amino-terminalDNA binding domain (DBD) and a carboxyl-terminal activation domain.LANA2 does not have conserved tryptophans in its amino-terminus requiredfor DNA-binding by IRF members, but has 32% amino acid identity over a71 bp region corresponding to the IRF4 interaction domain (IAD) (FIG.3A). Comparative phylogenetic analysis shows that the KSHV proteinsvIRF1, vIRF2 and LANA2 have a common branch point and appear to havearisen through gene duplication of a captured ancestral IRF-likecellular gene (FIG. 3B). In a previous study from our laboratorysurveying transcription of the KSHV genome in BC-1 cells (37), we failedto detect this transcript, possibly due to use of large probes coveringthis region which resulted in a low signal intensity on northernblotting.

LANA2 Expression In Vitro and In Vivo

Immunostaining using CM-10A2 mouse monoclonal antibody against LANA2 onKSHV infected cell lines (BC-1, BCBL-1, BCP-1) shows a fine granularnuclear pattern in all preparations (FIG. 4). This is similar to thesubnuclear distribution of LANA1 (ORF73) (11, 15, 23, 33). Doublestaining for LANA1 and LANA2 shows that the two proteins can co-localizeto some degree but that LANA2 has a much more diffuse pattern (FIG. 5).In mitotic cells, in which LANA1 bridges viral and cellular chromosomesto allow equal viral episome segregation, LANA1 aggregates with themitotic spindle (3). LANA2, however, is excluded from theseLANA1-containing mitotic figures suggesting that LANA2 unlike LANA1,does not play an important role in episome segregation during mitosis(FIG. 5).

Previous studies demonstrate that some genes (e.g. ORF K9) becomedysregulated in PEL tissue culture, and are expressed in established invitro cell lines but not parental PEL tumors. Other proteins, such asvIL-6, are expressed only in situ in a minority of PEL tumor cells (32).LANA2, in contrast, is expressed in virtually all KSHV infected cells inPEL and the majority of the KSHV infected cells in Castleman's diseasetumors (FIG. 6D, E and 7B). LANA2 is not appreciably expressed in KSspindle cells taken from skin biopsies (FIG. 6F). This is most clearlyseen in FIG. 7 in a lymph node containing both KS (endothelial cellorigin) and CD (B cell origin) tumors. LANA2 expression in this lymphnode occurs exclusively in the CD tumor cells but not KS spindle cells(FIG. 7B).

Lack of Seroreactivity to LANA2 in Serum from KSHV-Infected Patients

Unlike LANA1, LANA2 is unlikely to be a useful western blot antigen fordetecting KSHV antibodies. LANA2 expressed in COS7 cells failed to reacton western blotting with serum from patients with various KSHV-relateddisorders. None of 14 sera from individuals with AIDS-KS (n=4),classical KS (n=4), KSHV seropositive Castleman's disease (n=4) or PEL(n=2) showed serologic reactivity to LANA2 (FIG. 11). Negative controlsera from four blood donors (seronegative for ORF65 and LANA1 antigens)were also non-reactive, whereas the supernatants from two mousemonoclonal LANA2 hybridoma clones (CM-10A2 and CM-8B6) were positive. Wecannot exclude the possibility that other antigen formats (e.g.enzyme-linked immunoassay) might reveal a useful pattern for LANA2seroreactivity.

LANA2 Inhibits p53 Transactivation

Since LANA1 inhibits p53-mediated transcription and apoptosis (13), weexamined the effects of LANA2 on p53 function using the pG13-Lucpromoter reporter (containing 13 copies of the p53 response element)transiently transfected into SAOS-2 (p53 null) osteosarcoma cells.Transient expression of 0.5 mg p53 plasmid in SAOS-2 cells resulted inan 800-fold activation of the pG13-Luc reporter which was inhibited by87% on cotransfection of 0.5 mg pcDNA.LANA2 expression plasmid. Thistranscriptional repression was specific since the pGL-3 control promoteractivity (FIG. 8A) as well as the activity of a Gal4 reporter plasmid(not shown) was unaffected by pcDNA.LANA2 cotransfection. This effect isnot due to squelching since no transcriptional activation was seen atlow levels of LANA2 expression and increasing amounts of pcDNA.LANA2resulted in a monotonic repression of p53 activity on the pG13 reporter.

To determine if the same effect is present during endogenous p53activation, these experiments were repeated in U2OS cells (wild-type forp53) with and without treatment with 0.4 mM doxorubicin, achemotherapeutic agent which induces p53-mediated apoptosis. Doxorubicintreatment resulted in 13-fold activation of the pG13-Luc reporter andthis effect was inhibited 57% by 0.5 mg pcDNA.LANA2 transfection (FIG.8B).

LANA2 Protein—Protein Interactions

To determine if inhibition of p53 transactivation is due to directinteraction with p53 protein, we performed full length and truncatedGST-p53 pulldown assays using in vitro translated[³⁵S]-methionine-labeled LANA2. As seen in FIG. 9, GST-p53 fusionprotein precipitates LANA2 in vitro whereas no interaction is seen withGST protein alone. LANA2 interaction is localized to the region of p53comprising aa 290-393 and no interaction occurs with the truncated p53constructs containing aa 1-100 or aa100-290. In the reverse pull-downexperiments, GST-LANA2 but not GST alone showed specific interactionwith in vitro translated full length p53.

In vivo coimmunoprecipitation experiments, however, failed todemonstrate direct interaction between LANA2 and p53 (not shown). Inexperiments using naturally abundant p53 from BCBL-1 cells or SAOS-2cells in which p53 protein was overexpressed, no coimmunoprecipitationwas detected for LANA2 and p53 using either LANA2 (CM-10A2 and CM-8B6)or p53 (DO-1, Pab 1801, Ab-1) monoclonal antibodies. In part theseexperiments were inconclusive since we noted an unusual phenomenon inthat DO-1 (Santa Cruz), Pab 1801 (Santa Cruz) and Ab-1 (Oncogene)antibodies directed against p53 directly cross-react with LANA2. Thiswas confirmed by direct western blotting with these antibodies and thebacteria-derived GST-LANA protein in the absence of p53. We thus cannotexclude artifactual p53-LANA2 interactions in the GST-pulldown assays,or that antibody binding occurs at LANA2-p53 interaction site(s) whichinterfers with the immunoprecipitation reaction since the binding wasdone under native conditions. Yeast two-hybrid assays between LANA2 andfull-length p53 failed to clarify whether or not direct protein—proteininteractions occur in vivo (data not shown). LANA2 cloned into theGal4-BD cassette is toxic to the yeast and could not be evaluated. LANA2cloned into the Gal4-AD cassette and p53 into the Gal4-BD cassette,however, shows no interaction by b-galactosidase assay.

LANA2 Inhibits p53-Mediated Apoptosis

SAOS-2 cells are null for pRB as well as p53, and overexpression ofwild-type p53 in SAOS-2 cells results in apoptosis as indicated by thesubdiploid fraction (20%) of cells staining with propidium iodide in acell sorting profile (FIG. 10). In this experiment, cells werecotransfected with p53 and GFP expression plasmids, and DNA contentanalysis was performed only on cells gated for GFP. When LANA2 isexpressed together with p53 in SAOS-2 cells (FIG. 10C), a markeddiminution in subdiploid cells (from 20% to 10.8%) occurs indicating aspecific inhibition of p53-mediated apoptosis and genomic fragmentation.Similar results are obtained for U2OS cells, which have wild-type p53,treated with 0.4 uM doxorubicin for 30 hours, indicating that LANA2 caninhibit activation of endogenous p53 resulting from doxorubicintreatment (FIG. 10F). This was confirmed by caspase-8 activationfluorometric assays. Doxorubicin treated U2OS cells transfected withpcDNA.LANA2 showed lower levels of caspase-8 activation than doxoribicintreated U205 cells transfected with pcDNA empty vector control (data notshown).

C. Discussion

LANA2 is one of the few KSHV proteins which has been found to beexpressed in PEL and CD cells in vivo (11, 22, 32). However, unlikeLANA1, LANA2 is not expressed in the vast majority of KS spindle cells.These findings reinforce the concept that KSHV is capable of multiplelatency expression programs, and genes that are expressed in sometissues or cell lines may be silenced in others. LANA2 differs fromvIL-6, another KSHV protein whose protein expression is also limited toB cells, in that vIL6 is expressed in a minority population of PEL tumorcells. Since vIL-6 is a secreted cytokine, limited expression of vIL-6may nonetheless contribute to the pathogenesis of PEL tumors. Incontrast, LANA2 expression is uniformly present in PEL tumor cellsindicating that it too may have a critical role in maintaining the PELtumor cell phenotype. These patterns of expression could be expected ifthe vIL-6 promoter is activated by cytokine signaling pathways that aredependent on the local cellular milieu (unpublished observation, J.Osborne, Y. Chang, P. S. Moore), whereas the LANA2 promoter is activatedby B cell transcription factors.

KSHV is a gammaherpesvirus which, like EBV, has part of its naturallifecycle in CD19+ B-lymphocytes. It is apparent that a portion of theKSHV genome is devoted to maintenance of the virus in the B cellenvironment. B cells, for example, respond to antigen by activatingimmunoreceptor signaling pathways to achieve rapid clonal expansion.Under normal circumstances, induction of cell death by apoptosis occursafter B cell expansion to prevent lymphocytic hyperplasia (25). Theability of LANA2 to prevent p53-mediated B cell apoptosis would be anapparent benefit in maintaining an expanded population of infectedcells, or in preventing p53 pathway activation as part of a cellularantiviral response. While our in vitro studies suggest that LANA2inhibition of p53 activity is through direct protein—proteininteraction, caution is necessary in interpreting these results sincethey were not confirmable through in vivo interaction assays. The p53region binding LANA2 (aa. 290-393) in GST-pulldown assays includes thep53 tetramerization and regulatory domains, as well as residuesacetylated by p300 (17), suggesting a plausible mechanism.

The reasons why KSHV possesses two latency-expressed viral proteins,LANA1 and LANA2, to target the same p53 tumor suppressor protein areunclear. LANA1 is constitutively expressed in both KS lesions as well asKSHV-infected hematopoietic tissues and therefore appears to have abroader functional spectrum than LANA2. It is important to note that ourLANA2 experiments showing functional p53 inhibition were performed inosteosarcoma cell lines and so, at least under the conditions of ourassays, LANA2 inhibition of p53 is not unique to B cell lines.

Regardless of the mechanism for p53-inhibition, LANA2 is a likelycandidate protein involved in cell proliferation in hematopoietictissues. Inhibition of p53-induced apoptosis may contribute to B cellhyperplasia in Castleman's disease and to cell transformation in PELcells. Although KSHV vCYC is constitutively expressed on LT1 and LT2 inall infected cell lines, stable expression of this cyclin homolog hasbeen difficult to achieve in vitro since it induces apoptosis (31).Direct inhibition of both pRB and p53 signaling pathways by vCYCtogether with LANA1 and LANA2 could theoretically contribute toproliferative/neoplastic expansion of infected B cells.

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1. An isolated antibody which binds to an isolated polypeptidecomprising consecutive amino acids having the sequence set forth in SEQID NO:2, or a fragment thereof, encoded by a nucleic acid which encodesa Kaposi's sarcoma-associated herpesvirus latency-associated nuclearantigen 2 polypeptide or a fragment thereof.
 2. The antibody of claim 1,wherein the antibody is a monoclonal antibody.
 3. The antibody of claim1, wherein the antibody is a polyclonal antibody.
 4. The antibody ofclaim 1, wherein the antibody is humanized.
 5. The antibody of claim 1,wherein the antibody is detectable.
 6. The antibody of claim 5, whereinthe detectable antibody is labeled with a detectable marker.
 7. Thelabeled antibody of claim 6, wherein the detectable marker is aradioactive label or a colorimetric, or a luminescent, or a fluorescentmarker.
 8. A composition comprising the antibody of claim 1 and an agentconjugated to the antibody.
 9. The composition of claim 8 wherein theagent is a radioactive isotope or toxin.
 10. A composition comprisingthe antibody of claim 1 and a carrier.
 11. A method of treating asubject infected with Kaposi's sarcoma-associated herpesvirus, whichcomprises administering to the subject an amount of the composition ofclaim 10 under conditions such that the antibody binds to any Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide comprising consecutive amino acids having the sequence setforth in SEQ ID NO:2, or a fragment thereof, present in the subject, soas to thereby treat the subject.
 12. A method of determining whether asubject is afflicted with a disease associated with Kaposi'ssarcoma-associated herpesvirus infection of a B cell which comprises:(a) obtaining a suitable sample from the subject; (b) contacting thesuitable sample with the detectable antibody of claim 5 so as to form acomplex between the antibody and any Kaposi's sarcoma-associatedherpesvirus latency-associated nuclear antigen 2 polypeptide comprisingconsecutive amino acids having the sequence set forth in SEQ ID NO:2, ora fragment thereof, present in the sample; (c) removing any unboundantibody; and (d) detecting any antibody which is bound to any Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2 inthe sample, wherein the presence of bound antibody indicates that thesubject is afflicted with the disease.
 13. The method of claim 12,wherein the disease is Castleman's disease.
 14. The method of claim 12,wherein the disease is Primary Effusion Lymphoma.
 15. A method ofdetermining whether a subject is afflicted with a disease associatedwith Kaposi's sarcoma-associated herpesvirus infection of a B cell whichcomprises: (a) obtaining a suitable sample from the subject; (b)immobilizing an isolated capturing antibody to a support, wherein thecapturing antibody is capable of binding to Kaposi's sarcoma-associatedherpesvirus latency-associated nuclear antigen 2 polypeptide comprisingconsecutive amino acids having the sequence set forth in SEQ ID NO:2, ora fragment thereof; (c) removing any unbound capturing antibody; (d)contacting the immobilized capturing antibody with the suitable sampleso as to form a complex between the antibody and any Kaposi'ssarcoma-associated herpesvirus latency-associated nuclear antigen 2polypeptide or fragment thereof present in the sample; (e) removing anyunbound sample; (f) contacting any complex obtained in step (d) with thedetectable antibody of claim 5 so as to form a complex between thedetectable antibody and the complex; (g) removing any unbound detectableantibody; and (h) detecting any detectable antibody which is bound tothe complex wherein the presence of detectable antibody indicates thatthe subject is afflicted with the disease.
 16. The method of claim 12,wherein the disease is Castleman's disease.
 17. The method of claim 12,wherein the disease is Primary Effusion Lymphoma.